Single-cell RNA sequencing (scRNA-seq) was performed on 50-µm scrolls of formalin-fixed paraffin-embedded (FFPE) sagittal sections of mouse brain containing tumors confirmed by hematoxylin and eosin (H&E) staining on adjacent 5-µm sections.  The GEM-X Flex assay with 3' scRNA-seq prep type (10X Genomics) was used to generate cDNA libraries from FFPE tissues. 10,000 cells were targeted per sample.  Sequencing of cDNA libraries was carried out using an AVITI sequencer (Element Bio). 

Single-cell RNA-seq data from wild-type and truncated Ppm1d mice were processed in Seurat (1), as described previously (2).  Tumor cells were scored based on the similarity of their RNA-seq profiles to normal brain cell types from a normal brain atlas (3) as described previously (4).

References

(1) Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018; 36(5):411-420.

(2) Mangoli A, Valentine V, Maingi SM, et al. Disruption of ataxia telangiectasia-mutated kinase enhances radiation therapy efficacy in spatially directed diffuse midline glioma models. J Clin Invest. 2025; 135(12).

(3) Nowakowski TJ, Bhaduri A, Pollen AA, et al. Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex. Science. 2017; 358(6368):1318-1323.

(4) Reitman ZJ, Paolella BR, Bergthold G, et al. Mitogenic and progenitor gene programmes in single pilocytic astrocytoma cells. Nat Commun. 2019; 10(1):3731.